Phospholipase A2 (PLA2) is an enzyme with abundant pharmacological effects and unique mechanism of catalysis. This proposal is to study the quantitative structure-function relationship of PLA2 by a combination of genetic, biochemical, bioorganic, and biophysical techniques. Our system is PLA2 from bovine pancreas (14.7 kDa, 123 a.a.), overexpressed in E. coli. Specific Aim 1 focuses on the evaluation of the quantitative contribution of catalytic residues. In the previous period we have evaluated the contributions of Asp-99, Tyr-52, Tyr-73, Phe-22, and Phe-106. In this period we will continue to examine the roles of Asp-99 and His-48, and to try to use an exogenous amine or a single unnatural amino acid (e.g. N-methylhistidine at position 48) to replace the "catalytic diad" (Asp-99 ... His-48). The Specific Aim 2 is to identify critical residues involved in interfacial binding. Although crystallographers have suggested an "interfacial site" on the basis of crystal structures, enzyme molecules are not bound to an interface in the crystal. We propose to test a localized model suggested by P. Sigler and an extended model suggested earlier by B. Dijkstra. The importance of the N-terminal amino group will also be evaluated by unnatural analogues of alanine. Specific Aim 3 will tackle the quantitative contribution of the "hydrophobic channel" residues suggested by Sigler on the basis of the crystal structures. We plan to test this model by mutating Leu-2, Phe-5, IIe-9, Leu-19, and Tyr-69, and by introducing "superbulky" unnatural amino acids to block the entrance of the channel. Specific Aim 4 is to probe the structural and functional roles of the seven disulfide bonds. We plan to change each Cys/Cys pair to a double mutant Ala/Ala and examine the structural and functional perturbations in the mutants. The most important disulfides will be further changed to Cys/Homocys disulfides, as well as Lys/Asp and/or beta-aminoAla/Asp salt bridges. Specific Aim 5 is to convert the pancreatic PLA2 to mimic crotoxin and to examine the factors responsible for the neurotoxicity of crotoxin. In addition, the gene of crotoxin will be synthesized and overexpressed in E. coli, to be used by our collaborator M.-C. Tzeng for complementary structure-function studies. Specific Aim 6 will undertake the structural analysis of PLA2 and mutants by NMR at three levels: routine analysis of perturbations in mutant structures by 1D and 2D NMR (and by conformational stability determination); total analysis of the solution structures of WT PLA2 and its complex with the Gelb inhibitor by 2D, 3D, and 4D NMR; and analysis-of perturbations in mutant-inhibitor interactions by 2D NMR analysis of various mutant-inhibitor complexes. Inhibitors will be provided by M. Gelb.